bioRxiv preprint doi: https://doi.org/10.1101/034009; this version posted December 15, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Title

2 Evidence of adoption, monozygotic twinning, and low inbreeding rates in a large genetic

3 pedigree of polar bears

4

5 Authors

6 René M. Malenfanta,*, David W. Coltmana, Evan S. Richardsonb, Nicholas J. Lunnb, Ian

7 Stirlinga,b, Elizabeth Adamowicza, Corey S. Davisa

8

9 Affiliations

10 a Department of Biological Sciences, University of Alberta, Edmonton AB T6G 2E9, Canada

11 b Wildlife Research Division, Science and Technology Branch, Environment Canada, CW405

12 Biological Sciences Centre, University of Alberta, Edmonton AB T6G 2E9, Canada

13 * Corresponding author (email: [email protected], fax: 1–780–492–9234)

14

15

16 Note: Pre-review copy. A modified version of this article was accepted in Polar Biology on Dec.

17 15, 2015. DOI to be provided.

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18 Abstract

19 Multigenerational pedigrees have been developed for free-ranging populations of many

20 species, are frequently used to describe mating systems, and are used in studies of quantitative

21 genetics. Here, we document the development of a 4449-individual pedigree for the Western

22 Hudson Bay subpopulation of polar bears (Ursus maritimus), created from relationships inferred

23 from field and genetic data collected over six generations of bears sampled between 1966 and

24 2011. Microsatellite genotypes for 22–25 loci were obtained for 2945 individuals, and parentage

25 analysis was performed using the program FRANZ, including additional offspring–dam

26 associations known only from capture data. Parentage assignments for a subset of 859 individuals

27 were confirmed using an independent high-density set of single nucleotide polymorphisms. To

28 account for unsampled males in our population, we performed half-sib–full-sib analysis to

29 reconstruct males using the program COLONY, resulting in a final pedigree containing 2957

30 assigned maternities and 1861 assigned paternities with only one observed case of inbreeding

31 between close relatives. During genotyping, we identified two independently captured two-year-

32 old males with identical genotypes at all 25 loci, showing—for the first time—a case of

33 monozygotic twinning among polar bears. In addition, we documented six new cases of cub

34 adoption, which we attribute to cub misidentification or misdirected maternal care by a female

35 bereaved of her young. Importantly, none of these adoptions could be attributed to reduced

36 female vigilance caused by immobilization to facilitate scientific handling, as has previously been

37 suggested.

38

39 Keywords

40 Ursidae, Western Hudson Bay, alloparenting, relatedness, microsatellites, identical twins

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41 Introduction

42 Multigenerational pedigrees are useful in studies describing mating systems and for

43 quantitative genetics research (Pemberton 2008) and large pedigrees have been developed for

44 wild populations of many species, including red deer (Cervus elaphus; Slate et al. 2002), bighorn

45 sheep (Ovis canadensis; Poissant et al. 2010), song sparrows (Melospiza melodia; Reid et al.

46 2011), and American red squirrels (Tamiasciurus hudsonicus; Taylor et al. 2012). However,

47 because of the great effort and expense of sampling large carnivores, few pedigrees have been

48 developed for ursid species, with most parentage analyses containing no more than a few hundred

49 individuals, which are typically sampled non-invasively (Cronin et al. 2005; De Barba et al.

50 2010; Itoh et al. 2012; Onorato et al. 2004; however, cf. Proctor et al. 2004; Bellemain et al.

51 2006). To date, the largest parentage analysis of polar bears (Ursus maritimus) was based on 583

52 individuals in the Barents Sea (Zeyl et al. 2009a; Zeyl et al. 2009b), which showed that polar

53 bears exhibit serial monogamy, male-biased dispersal, and that inbreeding between close relatives

54 is rare.

55 Polar bears are large carnivores that occur at low densities throughout the circumpolar

56 Arctic and subarctic regions. They have a polygynous mating system (Derocher et al. 2010),

57 typically breeding between late March and June, with females giving birth to 1–3 cubs in

58 November–December while overwintering in maternity dens. Females emerge from dens in early

59 spring, and are the only providers of parental care until their cubs become independent—typically

60 at about 2.5 years old (Ramsay and Stirling 1988). Though family groups tend to avoid other

61 bears—perhaps to avoid cannibalism and other conspecific aggression (Taylor et al. 1985)—

62 cases of adoption have previously been documented (Atkinson et al. 1996; Belikov 1976;

63 Derocher and Wiig 1999; Lunn et al. 2000; Saunders 2005; Vibe 1976). Although adoption has

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64 been known to occur in the Barents Sea subpopulation (Derocher and Wiig 1999), Zeyl et al.

65 (2009a, 2009b) did not report any cases of adoption, perhaps because of the infrequency of

66 occurrence, combined with the study’s comparatively small sample size.

67 Adoption has been observed in more than 60 mammalian species (Gorrell et al. 2010), and

68 its occurrence requires special explanation due to the extremely high cost of milk provision to

69 adopted young (e.g., Clutton-Brock et al. 1989). Allonursing and adoption may be explained

70 adaptively through kin selection, reciprocal altruism, evacuation of excess milk, or through a gain

71 in parenting experience (Roulin 2002). Alternately, adoption and allonursing may simply be the

72 result of error, occurring especially when a reproductive individual is already hormonally or

73 behaviourally primed to provide parental care and is bereaved of their young (Riedman 1982).

74 Most empirical studies support the kin selection, milk evacuation, or misdirected parental care

75 hypotheses (Roulin 2002). Amongst polar bears, adoption has been attributed to misdirected

76 parental care caused by cub misidentification (Lunn et al. 2000), which may be caused by

77 confusion due to the immobilization of adult females, which is necessary for scientific handling

78 (Derocher and Wiig 1999).

79 Like adoption, monozygotic twinning is taxonomically widespread but infrequent, and

80 although well described in humans (e.g., Bulmer 1970) and cattle (e.g., Silva del Río et al. 2006),

81 few cases have been documented in wildlife species. Monozygotic quadruplets are the normal

82 mode of reproduction among some species of armadillos (Hardy 1995), and monozygotic twins

83 have been identified in lesser flat-headed bats (Tylonycteris pachypus; Hua et al. 2011), in wolves

84 (Canis lupus; Carmichael et al. 2009), among some species of pinnipeds (Spotte 1982), including

85 Antarctic fur seals (Arctocephalus gazella; Hoffman and Forcada 2009), and possibly in mule

86 deer (Anderson and Wallmo 1984). The apparent scarcity of monozygotic twins is partially

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87 attributable to the difficulty of identifying them, as this requires genetic or embryological

88 confirmation. For instance, conjoined twins, which develop from monozygotic twins and are

89 therefore far rarer, are phenotypically conspicuous, and at least 20 cases of conjoined twinning in

90 wildlife species have been published (Kompanje and Hermans 2008). To our knowledge,

91 identical twinning (or conjoined twinning) has never been detected in any species of bear,

92 although no studies have had large enough sample sizes to reliably detect such a rare event.

93 In this paper, we present a large pedigree of polar bears comprising 4449 individuals from

94 the Western Hudson Bay subpopulation captured over six bear generations in northeastern

95 Manitoba, Canada between 1966 and 2011. We document six new cases of cub adoption, and

96 show—for the first time—an instance of monozygotic twinning among polar bears. Further, we

97 find no cases of inbreeding between first-degree relatives. This pedigree is now being used to

98 determine the mating system of polar bears (Richardson 2014), and in future studies, this

99 pedigree will be used to determine the heritabilities of various body size metrics, some of which

100 have been declining in this subpopulation for decades (Stirling and Derocher 2012).

101

102 Methods

103 Sample collection

104 Most tissue samples were collected from bears that were immobilized and handled as part

105 of long-term ecological studies of polar bears in Western Hudson Bay. However, a small number

106 of samples were collected from bears captured by Manitoba Conservation staff near the

107 community of Churchill as part of the Polar Bear Alert Program (Kearney 1989) or from polar

108 bears harvested each year as part of a legal, regulated subsistence hunt by Inuit living along the

109 coast of western Hudson Bay (Derocher et al. 1997; Taylor et al. 2008). Sampling locations are

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110 shown in Figure 1. During the first handling of each captured individual, he/she was assigned a

111 unique ID applied as a permanent tattoo on the inside of the upper lip and affixed as a plastic tag

112 in each ear. Skin samples were collected by retaining leftover pinnal tissue from ear-tagging or

113 from adipose tissue samples collected using a 6-mm biopsy punch of superficial fat on the rump

114 (Ramsay et al. 1992; Thiemann et al. 2008). A temporary paint mark is also applied to avoid

115 recapture of the same individual twice in one season. Blood samples were collected by drawing

116 blood from a femoral vein into a sterile vacutainer. All samples were stored at –80°C until DNA

117 extraction. If the age of a newly sampled individual was unknown (i.e., not a cub-of-the-year or a

118 dependent yearling), a vestigial premolar tooth was extracted for age determination using

119 measurement of cementum annulus deposition (Calvert and Ramsay 1998). All individuals

120 handled by Environment Canada were sampled in autumn and were selected for handling

121 indiscriminately of age or sex; however, in every year from 1980 onward (except for 1985 and

122 1986), a springtime sampling effort was also included, in which only adult females and their

123 cubs-of-the-year—which had recently emerged from maternity dens—were handled.

124

125 DNA extraction and microsatellite genotyping

126 Total genomic DNA was extracted from fat, skin, or leukocytes recovered from ACK-

127 lysed blood using DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). We genotyped 2945

128 individuals born between 1960 and 2011, including duplicate samples from 69 individuals

129 included to estimate genotyping error rates. Individuals born before 2006 were genotyped at all

130 25 microsatellite loci (Table S1, Supplementary Material); however, because of changes to the

131 genotyping protocol made in 2012 to streamline microsatellite multiplexing, individuals born

132 from 2006 onward were genotyped at 24 loci (excluding CXX173). PCR products from

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133 microsatellite amplifications were resolved on an Applied Biosystems 377 DNA Sequencer,

134 3100-Avant DNA Analyzer, or a 3730 DNA Analyzer, and sized relative to Genescan size

135 standards. Genotyping was performed using the programs Genotyper and Genemapper (Applied

136 Biosystems, Foster City, CA, USA).

137

138 Genetic diversity, tests of disequilibrium, and statistics

139 The number of observed alleles (NA), observed heterozygosities (HO), expected

140 heterozygosities (HE), probabilities of exclusion (Pex), and probabilities of identity (PID) were

141 calculated using GENALEX 6.5 (Peakall and Smouse 2006; Peakall and Smouse 2012); Pex was

142 calculated using the formula for single unknown parent exclusion from Jamieson and Taylor

143 (1997). Departures from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD)

144 were assessed with exact tests (Guo and Thompson 1992) and a Markov chain (dememorization

145 number = 5000, number of batches = 1000, number of iterations per batch = 2000) using

146 GENEPOP ON THE WEB 4.2 (Raymond and Rousset 1995; Rousset 2008). The total genotyping

147 error rate (calculated as the sum of the allelic dropout rate (E1) and false allele rate (E2), Table

148 S1) was calculated from duplicate samples using the program PEDANT 1.0 (Johnson and Haydon

149 2007) using 100,000 replicates. Unless otherwise indicated, all other statistics were calculated in

150 R 3.1.3 (R Core Team 2015), and a significance level of α=0.05 was used for all tests.

151

152 Pedigree generation and adoption detection

153 We used the program FRANZ 2.0 (Riester et al. 2009; Riester et al. 2010) to generate an

154 initial pedigree from our microsatellite data, using a sub-pedigree comprising known mother–cub

155 relations from field data, individuals’ years of birth (and death, if known) and the settings

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156 specified in Table 1. FRANZ generates a multigenerational pedigree in a single step, using

157 simulations to determine expected parent–offspring mismatch rates, simulated annealing to

158 estimate the maximum-likelihood pedigree, and Metropolis-coupled Markov chain Monte Carlo

159 sampling to calculate parentage posterior probabilities. As part of its simulation step, FRANZ

160 uses the empirical distribution of parent–offspring mismatch rates to identify problematic

161 parental assignments in the sub-pedigree (Figure 2), which we have classified here as adoptions.

162 Although our field records contain no instances of females younger than four years old (or older

163 than 28 years old) having successfully given birth, we specified a possible reproductive age range

164 of 2–32 (at time of parturition) to account for outliers amongst unobserved maternities as well as

165 possible errors in tooth aging. For simplicity, we also used this same age range for males; males

166 generally do not become reproductively active until at least their fifth or sixth year, though they

167 may begin to produce spermatozoa at age two (Rosing-Asvid et al. 2002). The oldest female and

168 male polar bears captured from the Western Hudson Bay subpopulation were 32 and 29 years

169 old, respectively.

170 To detect the genetic mothers of adopted individuals, we removed their links from our

171 field-data sub-pedigree and re-ran FRANZ. Then, to validate and error-correct the resultant

172 pedigree, we used the program VIPER 1.01 (Paterson et al. 2012) to examine the inheritance of

173 4,475 single-nucleotide polymorphisms (SNPs) genotyped in a 859-individual subset of

174 pedigreed bears. These SNPs were developed from transcriptomic and RAD sequencing and were

175 genotyped with high fidelity in all individuals using a recently developed 9K Illumina BeadChip

176 for polar bears (Malenfant et al. 2015). We removed all pedigree links displaying more than one

177 SNP inheritance error, which we determined as a cutoff based on the empirical distribution of

178 inheritance errors.

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179 Finally, to account for a lower proportion of males than females being sampled in our data

180 (which led to proportionally fewer paternity than maternity assignments), we used the program

181 COLONY 2.0 (Jones and Wang 2010) to generate hypothetical sires and differentiate between

182 full siblings and maternal half siblings. To reduce false paternity assignments, we limited

183 candidate offspring in this analysis to 760 individuals having unassigned sires but genetically

184 assigned dams. All individuals were pooled in a single analysis irrespective of birth year to allow

185 for the possibility that a hypothetical male had sired multiple offspring across years. We allowed

186 for male and female polygamy, using maternal and paternal sibship priors of 3.655 and 2.968

187 respectively, which were determined empirically from the pedigree. All pedigree statistics were

188 calculated using the package PEDANTICS 1.5 (Morrissey and Wilson 2010).

189

190 Genetic relatedness

191 Asocial animals such as polar bears might choose to adopt nearby orphans if they are

192 genetically related, as this can provide an inclusive fitness advantage to the foster parent (e.g.,

193 Gorrell et al. 2010). To determine if adopted cubs were genetically related to their foster mothers,

194 we used the program COANCESTRY 1.0.1.2 (Wang 2011) to obtain the Queller-and-Goodnight

195 (1989), Lynch-and-Ritland (1999), and Wang (2002) relatedness metrics using allele frequencies

196 and error rates estimated from the full microsatellite dataset. Because all estimators gave similar

197 results, only the Queller-and-Goodnight estimator results are presented. This estimator was

198 designed for studies of kin selection and has the property that unrelated individuals are expected,

199 on average, to have a relatedness of zero.

200

201 Results

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202 Microsatellite genotypes

203 Complete 25-locus genotypes were obtained for 2418 individuals, 24-locus genotypes

204 were obtained for 478 individuals, 23-locus genotypes were obtained for 34 individuals, and 22-

205 locus genotypes were obtained for 15 individuals. The mean number of observed alleles (NA) was

206 7.6 (range: 3–10), and mean observed (HO) and expected (HE) heterozygosities were both 0.672

207 (ranges: 0.112–0.847 and 0.112–0.840, respectively). Two loci, G1A and G10L, deviated from

208 HWE (PG1A = 0.0028, PG10L = 0.0064) but were not significantly out of HWE following a strict

209 Bonferroni correction for multiple tests (αcorrected = 0.002). Complete summary statistics for

210 microsatellite loci are presented in Table S1. Thirty pairwise tests of LD were significant

211 following strict Bonferroni correction, however, this was likely because our dataset contained

212 many groups of related individuals. Combined probability of exclusion (Pex) over all loci is

213 0.99991; this drops to 0.99988 if CXX173 is excluded. Combined probability of identity (PID)

214 was 7.102×10−23 for unrelated individuals and 1.562×10−9 for full siblings; these increase to

215 5.020×10−22 and 3.551×10−9 respectively if CXX173 is excluded. Total genotyping error rate was

216 estimated at 0.36%. Fourteen successfully genotyped individuals were removed prior to pedigree

217 generation because their year of birth was unknown.

218

219 Pedigree statistics and inbreeding

220 We supplemented the remaining 2931 genotyped-and-aged individuals with 1225

221 individuals known only from field observation. FRANZ assigned 2972 maternities using field

222 and/or genetic data and 1105 paternities using genetic data alone. Based on SNP inheritance

223 errors identified in VIPER, 4 offspring–sire links out of 163 (≈2.5%) and 15 offspring–dam links

224 out of 465 (≈3.2%) were removed. In all 15 of these cases, offspring–dam relationships had been

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225 inferred using genetic data only (i.e., they were not based on field observations). COLONY

226 reconstructed 293 sires, which collectively accounted for 760 paternal assignments (mean ± SD

227 offspring per reconstructed sire = 2.6 ± 1.4), and brought the pedigree to 4449 individuals in

228 total. Nine females aged 3 years or younger and males aged 2 years or younger (at time of

229 conception) were assigned as parents in the final version of the pedigree, though the ages of 17 of

230 these 18 individuals were uncertain as they were derived from tooth-aging estimates. The oldest

231 dam and sire assigned in the pedigree were 28 and 30 respectively at the time of conception.

232 Including COLONY-reconstructed individuals, the pedigree contains 1381 founders (i.e.,

233 individuals of unknown parentage) and extends to six generations for some individuals. Of 382

234 mating events in which the identities of both parents and at least one grandparent on each side

235 were known (a necessity for detecting inbreeding), only three individuals had non-zero

236 inbreeding coefficients: X11088, X11089, and X11389. X11088 and X11089 are littermates born

237 in 1989 to X10668 after mating with her half-brother X10497; X11389 was supposedly born to

238 X09396 and her brother X09398 (however, cf. the Discussion regarding this mating). A graphical

239 view of the pedigree and complete pedigree statistics and are given in Figure 3 and Table S2 of

240 the Supplementary Material, respectively.

241

242 Monozygotic twinning

243 We detected one pair of identical twins among 574 genotyped twin litters and 37

244 genotyped triplet litters: cubs X17324 and X17326 match at all 25 loci (Supplementary Material

245 2). Both individuals were independent two-year olds at the time of capture (November 10 and 11,

246 2003, respectively), and were handled ~3.5 km apart on opposite sides of the Churchill airport.

247 They were known not to have been recaptures of the same individual because the second-

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248 captured individual (X17326) lacked the temporary paint mark and did not have a permanent

249 tattoo or ear tags. If dizygotic, the probability of these cubs sharing a genotype at all 25 loci is

250 1.64×10–11, as calculated from the full genotypes of both parents. To discount the possibility that

251 these identical genotypes were the result of sample mix-up, we reconfirmed the genotype of

252 X17324 using a second, independently collected tissue sample; unfortunately, a second sample

253 for X17326 was not available. However, because these individuals were handled on different

254 days, the probability of sample mix-up during fieldwork is extremely low.

255

256 Cases of adoption

257 We identified six previously undetected cases of adoption occurring between 1981 and

258 2004 and identified four of the six genetic mothers (Table 2, Supplementary Material S2). In five

259 of these cases, cubs were adopted during their first year of life; in the remaining case (X09059), it

260 was unclear if the cub was adopted during its first or second year. In two cases, adoptive mothers

261 were observed to have fostered cubs for at least a year, and from later capture and harvest

262 records, it is known that at least five of six adopted cubs survived to independence (though the

263 fate of X11097 is unknown.) Although five of the six adopted cubs were female, there was no

264 statistical evidence of preference to adopt females over males (binomial test of 1:1 ratio: P =

265 0.2188). All adopted cubs appeared to be unrelated to their adoptive mothers: average adoptee–

266 adopter relatedness is −0.038, and 95% confidence intervals for the Queller-and-Goodnight

267 relatedness estimators are not significantly different from 0 in all cases. In two of the six adoption

268 cases, females were also accompanied by their own genetic offspring.

269

270 Discussion

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271 Inbreeding

272 Active inbreeding avoidance is often presumed to be common amongst animals because

273 of reduced fitness of inbred offspring (Keller and Waller 2002), though tolerance of—or even

274 preference for—inbreeding may occur because of inclusive fitness benefits (Szulkin et al. 2013).

275 When inbreeding avoidance does occur, it is generally attributed to mate choice or sex-biased

276 dispersal (Pusey and Wolf 1996). However, sex-biased dispersal may also occur for reasons

277 unrelated to inbreeding, such as sex differences in the benefits of retaining a productive territory

278 or avoidance of intersexual competition (Moore and Ali 1984). Little is known about inbreeding

279 in polar bears, and primary among the motivations for developing this pedigree was the

280 characterization of inbreeding in this subpopulation (Richardson et al. 2006).

281 We detected only two instances of incestuous mating: one between half-siblings X10668

282 and X10497 (producing X11088 and X11089), and another putative case between full-siblings

283 X09396 and X09398 (producing X11389). However, in this latter case, X09398 is almost

284 certainly a false paternity assignment: X09396 is an ungenotyped dam that was assigned using

285 only field data, causing X09398 to be incorrectly assigned as a father because of allele-sharing

286 with his sister. Therefore, after excluding this case, inbreeding among close relatives appears to

287 be extremely rare in the Western Hudson Bay subpopulation, occurring only once among 382

288 mating events in which it could have been observed. For comparison, in a study of the Barents

289 Sea subpopulation, a single instance of father–daughter inbreeding was detected amongst 22

290 matings between parents of known identity (Zeyl et al. 2009a), suggesting that the rate of mating

291 between first-degree relatives was ~4.5%.

292 Polar bears exhibit low variation at major histocompatibility loci (Weber et al. 2013),

293 which are thought to play an important role in kin recognition (Villinger and Waldman 2012),

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294 and it has also been suggested that polar bears have undergone little selection for kin recognition

295 because of low population densities (Lunn et al. 2000). If this is the case, then mate choice is

296 unlikely to explain low inbreeding rates amongst polar bears. In contrast, our finding of little-to-

297 no inbreeding in the Western Hudson Bay subpopulation may result from substantial dispersal

298 and interbreeding between Western Hudson Bay and adjacent management units (Richardson

299 2014). Studies of American black bears (Ursus americanus; Costello et al. 2008) and brown

300 bears (U. arctos; Bellemain et al. 2006) have found similar rates of close inbreeding, which were

301 attributed to lack of opportunity resulting from low population density and male-biased dispersal.

302 In another study, inbreeding avoidance was cited as the most likely cause of male natal dispersal

303 among brown bears (Zedrosser et al. 2007). These findings are also likely to hold true for polar

304 bears, which occur at even lower densities, and for which limited genetic evidence also suggests

305 male-biased dispersal in some subpopulations (Zeyl et al. 2009b). However, because radio

306 telemetry data for male polar bears is scarce, little is known about dispersal patterns in male polar

307 bears and further study is needed.

308

309 Monozygotic twinning

310 Inclusive fitness theory predicts the possible spread of genes for monozygotic twinning

311 (Gleeson et al. 1994; Williams 1975), and though the reason for the rarity of monozygotic

312 twinning is not well understood, it may be partially attributable to higher rates of spontaneous

313 abortion for monozygotic twins (Livingston and Poland 1980) and lower survival of twins in

314 species that normally bear only one offspring (e.g., Fricke 2001). Based on observed sex ratios of

315 multi-cub litters, Ramsay and Stirling (1988) determined that monozygotic twinning was likely

316 rare or absent among polar bears. Our study confirms that monozygotic polar bear twins are

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317 extremely rare, being found in less than 1/600 litters (0.17%) in our data. To our knowledge,

318 this is the first confirmed record of monozygotic twinning among polar bears or any other ursid,

319 and previous genetics studies of bears (e.g., Bellemain et al. 2006; Proctor et al. 2004; Zeyl et al.

320 2009a) likely failed to detect twins because of smaller sample sizes. Slightly higher rates of

321 monozygotic twinning have been found for humans (0.35–0.4%; Bulmer 1970) and for cattle

322 (0.33%; Silva del Río et al. 2006). In part, the lower estimate for polar bears may result from

323 discounting the “invisible fraction” (Grafen 1988) of identical twins that were never observed

324 because at least one cub died prior to emergence from the maternity den.

325

326 Adoption

327 According to Hamilton’s (1964) theory of kin selection, natural selection will favour a

328 heritable predisposition for altruistic behaviour when C < rB, where C is the fitness cost to the

329 altruist, B is the fitness benefit to recipient, and r is the relatedness between these individuals.

330 Thus, kin selection requires greater-than-average relatedness between altruist and recipient, and

331 relatedness must be particularly high to account for such energetically costly behaviours as

332 adoption and nursing. Lunn et al. (2000) ruled out kin selection as an explanation for three

333 previous cases of polar bear adoption based on low genetic relatedness. Our results reinforce the

334 finding that adopted cubs and their foster mothers are unrelated, and that kin selection does not

335 appear to drive adoption in this population. Though reciprocal care of offspring has been

336 observed in polar bears (Lunn 1986), given the polar bear’s low population density and generally

337 asocial nature, reciprocal altruism is also extremely unlikely, and no reciprocal cases of adoption

338 were observed in our data.

15 bioRxiv preprint doi: https://doi.org/10.1101/034009; this version posted December 15, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

339 Milk evacuation may explain allonursing behaviour in some pinnipeds (Riedman and

340 Boeuf 1982) and bats (Wilkinson 1992), however, it is extremely unlikely to explain adoption in

341 polar bears. Whereas it is beneficial for pinnipeds and bats to be leaner to increase diving or

342 flight efficiency (Roulin 2002), lean polar bears lack the energy storage and thermal benefits

343 (Pond et al. 1992), as well as the reproductive benefits (Stirling et al. 1999) associated with body

344 condition. This is particularly true in the Western Hudson Bay subpopulation, where mothers

345 may fast for four or more months during the ice-free period each year. In this subpopulation, a

346 female’s ability to maintain pregnancy (Derocher et al. 1992) and the survival of her own cubs

347 (Derocher and Stirling 1996) are mass-dependent so that a female would gain no apparent benefit

348 from milk evacuation. Since it appears that all six foster mothers had birthed genetic litters by the

349 time of adoption, the parental experience hypothesis is also unlikely to account for any of these

350 adoptions (Roulin 2002).

351 Adopted cubs were captured alone with their foster mother in four of six cases, and in all

352 these cases, the adopted cub is known to have survived to independence, implying the

353 provisioning of milk by the mother to the adoptive offspring, as has been observed directly in at

354 least once instance of fostering (Belikov 1976). Because spontaneous lactation is not believed to

355 occur in most species and has only been consistently demonstrated among dwarf mongooses

356 (Helogale parvula) suckling close relatives (Creel et al. 1991), it is highly unlikely to explain

357 allonursing of alien offspring among polar bears. This suggests that these cubs’ adoptions

358 coincided with the loss of the females’ biological litters (either due to death or because of

359 unintentional cub-swapping with another female), while females were biologically capable of

360 suckling. In our remaining two adoption cases, cub misidentification is the most likely

361 explanation, as adopted cubs were accompanied by the female’s own biological offspring. Cub

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362 mixing sometimes occurs in both polar bears and brown bears (Glenn et al. 1976; Lunn 1986),

363 and in any of these adoption cases, cub-swapping may have occurred due to simple

364 misidentification during periods of high bear density, such as springtime den emergence or the

365 autumn fasting period ashore (Derocher and Stirling 1990; Ramsay and Stirling 1988). However,

366 we note that in at least one previously observed case of adoption, it has been proposed that a

367 female with two cubs of her own adopted two of another female’s cubs she was killed in a fight

368 (Vibe 1976), and cubs may also become separated from their mothers if males drive them off in

369 order to mate with her (I. Stirling, unpublished data).

370 It has been suggested that scientific handling may increase the probability of cub

371 abandonment or adoption if maternal vigilance is reduced during the time it takes to fully recover

372 from immobilization (Derocher and Wiig 1999). Importantly, we found no evidence to support

373 this hypothesis. We were able to identify four of the six genetic mothers, two of which had not

374 been captured for five years prior to the adoption, and the remaining two of which were not

375 captured until after the adoption. Likewise, none of the six foster mothers was captured in the

376 period between the adopted cub’s birth and their first observation together. This finding

377 corresponds with a number of studies that have failed to find a significant negative correlation

378 between scientific handling and litter size (Amstrup 1993; Lunn et al. 2004), cub survival

379 (Ramsay and Stirling 1986; Rode et al. 2014), or on the cohesion of family groups (Messier

380 2000).

381

382 Acknowledgments

383 The authors would like to thank the Manitoba Department of Conservation and the

384 Government of Nunavut for providing some of the samples, as well as Dennis Andriashek and

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385 Wendy Calvert for long-term data collection and maintenance. We would also like to

386 acknowledge the contributions of Andrew Derocher and the late Malcolm Ramsay, who gathered

387 some of the field data used in this study. DNA extractions for most pre-2006 tissue samples were

388 conducted by Jennifer Bonneville Davis. This project was funded by grants to CSD from

389 Environment Canada and by a Natural Sciences and Engineering Research Council Discovery

390 Grant to DWC (Grant ID 312207-2011). RMM is funded by scholarships from Alberta Innovates

391 Technology Futures, the University of Alberta, and the Province of Alberta. Financial and

392 logistical support of the long-term study of polar bears in western Hudson Bay have been

393 provided by Care for the Wild International, the Churchill Northern Studies Centre, Environment

394 Canada, the Isdell Family Foundation, Manitoba Conservation, Natural Sciences and Engineering

395 Research Council, Nunavut Wildlife Research Trust Fund, Parks Canada Agency, the Strategic

396 Technology Applications of Genomics in the Environment (STAGE) funding program, Wildlife

397 Media Inc., World Wildlife Fund (Canada), and World Wildlife Fund Arctic Programme. Josh

398 Miller and Jamie Gorrell provided advice and comments on an earlier version of the manuscript.

399

400 Ethical standards

401 All applicable international, national, and/or institutional guidelines for the care and use

402 of animals were followed. Environment Canada's animal-handling procedures were approved

403 annually by their Prairie and Northern Region Animal Care Committee, and all research was

404 conducted under wildlife research permits issued by the Province of Manitoba and by Parks

405 Canada Agency.

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406 References

407

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24 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable

635 Table 1. Non-default settings used for pedigree generation in the program FRANZ.

Parameter Setting doi: Female reproductive age range 2–32 https://doi.org/10.1101/034009 Male reproductive age range 2–32 Maximum number of candidate mothers 5,000 Maximum number of candidate fathers 5,000 Number of simulation iterations 1,000,000 Maximum number of simulated annealing iterations 1,000,000,000 Number of Metropolis–Hastings burn-in iterations 5,000,000

Number of Metropolis–Hastings iterations 30,000,000 under a

Metropolis–Hastings sampling frequency 100 ; this versionpostedDecember15,2015.

636 CC-BY-NC-ND 4.0Internationallicense The copyrightholderforthispreprint(whichwas .

25 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable

637 Table 2. New cases of polar bear adoption reported in this paper. PM = proportion of loci mismatched between cub and candidate

638 mother; rQG = Queller-and-Goodnight relatedness between cub and the adoptive mother. In two cases (represented as “—”), the true doi: 639 dam could not be determined. Asterisks denote individuals whose genotypes could be confirmed by genetic assignment of their other https://doi.org/10.1101/034009 640 observed offspring to them; most cubs’ genotypes could not be confirmed in this manner because they are not known to have parented 641 offspring. Year of Observed dam Date(s) observed Age of Other cubs r Inferred dam Survived to Cub Sex QG birth (PM) together observed dam in litter (95% CI) (PM) independence X05562* −0.19 X09913* X09059* Female 1980 Aug. 10, 1981 7 0 Yes (10/25) (−0.36 – 0.03) (0/25) * X10607 −0.05 under a X10608 Female 1987 Sept. 24, 1987 8 0 — Yes

(5/25) (−0.22 – 0.12) ; * * this versionpostedDecember15,2015. X05668 −0.07 X11456 CC-BY-NC-ND 4.0Internationallicense X11097 Female 1989 Mar. 22, 1989 6 2 Unknown (9/25) (−0.35 – 0.14) (0/25) X03318* 0.17 X17069 Male 1998 Nov. 30, 1998 24 0 — Yes (4/25) (0.00 – 0.36) X17082* Sept. 22, 2003 – 0.03 X10688* X17294* Female 2003 7–8 1 Yes (4/25) Nov. 8, 2004 (−0.12 – 0.21) (0/25) X11940* Sept. 18, 2004 – −0.12 X12273* X19939 Female 2004 11–12 0 Yes (12/25) Sept. 18, 2005 (−0.38 – 0.10) (1/25) 642 The copyrightholderforthispreprint(whichwas .

26 bioRxiv preprint doi: https://doi.org/10.1101/034009; this version posted December 15, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

643 644 Figure 1. Sampling locations of bears included in the Western Hudson Bay pedigree. 645 Imagery ©2015 TerraMetrics, Inc. (www.terrametrics.com), accessed via Google Maps and 646 ggmap 2.4 (Kahle and Wickham 2013). 647

27 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable doi: https://doi.org/10.1101/034009 under a ; this versionpostedDecember15,2015. CC-BY-NC-ND 4.0Internationallicense The copyrightholderforthispreprint(whichwas .

648 649 Figure 2. Number of mismatched microsatellite loci for simulated and actual parentage assignments. Simulation results represent 650 2,000,000 parent–offspring pairs generated in FRANZ using empirically estimated error rates. Asterisks indicate putatively adopted 651 individuals.

28 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable doi: https://doi.org/10.1101/034009 under a ; this versionpostedDecember15,2015. CC-BY-NC-ND 4.0Internationallicense The copyrightholderforthispreprint(whichwas .

652 Figure 3. Graphical representation of the 4449-individual polar bear pedigree described in this paper. Each point is an individual bear. 653 Maternities are represented by red lines; paternities are represented by blue lines.

29